Group #

Name: Professor:
Date:

Activity 2
Estimation of Phosphate Content in Soft
Drinks

I. Introduction:

Principle of Colourimetry:

A colourimeter can be used to measure any test substance that is itself coloured or can be reacted to produce
a colour.

It is a device consisting of,

1) A light source,
2) Some optics for focusing the light,
3) A coloured filter, which passes light of the colour which is absorbed by the treated sample,
4) A sample compartment to hold a transparent tube or cell containing the sample and
5) A light-sensitive detector.

Simplified diagram of a colourimeter

Beer-Lambert Law:

The Beer-Lambert Law relates the absorption of light at a particular wavelength to the concentration of the
substance that is absorbing the light as follows:

Where,  A is the absorption at a particular wavelength.

 ε is the molar absorption coefficient for the compound at the wavelength at which the absorption is
measured and is characteristic for the compound. This constant is a measure of how strongly a
compound absorbs at a particular wavelength.
 c is the concentration of the compound and is independent of wavelength.
 l is the path length of the test tube used to hold the sample.

To estimate the amount of phosphate in soft drink sample, one first constructs a graph from stock solutions of
phosphate. The absorbance associated with a set of phosphate solutions of known concentrations is called a
phosphate standard curve. The amount of phosphate in sample solution can be determined from the standard
curve by drawing a horizontal line on the graph parallel to the X-axis which corresponds to the absorbance of
soft drink sample. This line will intersect the standard curve; at this intersection, a vertical line is drawn to the
X-axis. This point corresponds to the volume of phosphate in sample solution.

II. Objectives
 To determine the amount of phosphate in soft drinks

III. Procedure:

Preparation of Solutions:

A. Molybdate Solution:
 Add 13.6 mL of con.sulphuric acid to 35 mL of water and allow the solution to cool. In a separate vessel
add 2.5 g of ammonium molybdate to 50 mL of water. Add sulphuric acid solution to it & make up the
volume.

B. Reducing Solution:
 Dissolve 11.25 g sodium hydrogen phosphate in 30 mL water. Add 2.25 g sodium sulphate and 0.19 g 1-
amino 2-naphthol 4-sulphonic acid and dilute the mixture to 75 mL with water. Stir the mixture
thoroughly & filter.

C. Standard Solutions of Phosphate:

 Dissolve 20 mg of potassium dihydrogen phosphate in 100 mL of water. The concentration of phosphate
standard solution is 0.2 mg/mL.
 Prepare 9 standard solutions containing 0.5 mL molybdate solution, 0.2 mL reducing solution & x mL of
phosphate stock solution + y mL of distilled water. Where, x+y =9.3 mL and x= 2-0.2n,
n = 1,2,…......... 9.
 Also prepare a blank solution by mixing 0.5 ml of molybdate solution, 0.2 ml of reducing solution and
9.3 mL of water.
  Set the wave length to 635 nm and adjust the colorimeter reading to zero with the blank.
 Place each standard solution of phosphate in turn in the curette and record the absorbance.
 Dilute the soft drink sample 5 times. Mix 1mL of this diluted solution with 0.5 mL molybdate, 0.2 mL
reducing solution and 8.3 mL water, so as to get the test solution.
 Measure the absorbance of the sample solutions.
 Plot a graph between absorbance and volume of phosphate in various standard solutions and obtain the
calibrated curve.
 From the curve determine the amount of phosphate in the test solution.

D. Calculations:
The amount of phosphate in the sample of soft drink is 5 x 0.2 x V = 1 x V mg /mL.
5 is the dilution factor.
0.2 mg/mL amount of phosphate in standard solution.
V is the volume of phosphate solution obtained from graph.

IV. Data and Results

Sample Absorbance (T1) Absorbance (T2)

Blank
0.2 mL
0.4 mL
0.6 mL
0.8 mL
1.0 mL
1.2 mL
1.4 mL
1.6 mL
1.8 mL
Sample1
Sample 2

V. Calculations:

VI. Documentations:

VII. Conclusion:

VIII. Reference/s: